1.What is the full-form of the HPLC?
Answer: The full-form of HPLC is high performance liquid chromatography or high-pressure liquid chromatography
2. What is the principle of HPLC?
HPLC (high-pressure/high-performance liquid chromatography) is a separation technique based on solid stationary and liquid mobile phases.
Separations depend upon the polarity of the stationary phase, mobile phase and molecules. Separation may be achieved by either the Partition mechanism or the Adsorption mechanism
3. What are the different components of HPLC?
The following are the different components of HPLC:
- Mobile Phase
- Mobile phase reservoir
- Degassers
- Pump
- Mixing valve
- Guard column
- Sample injection port
- Injector
- Column
- Column temperature controller
- Detector
- Waste Collector
- Data processor &
- Chromatogram
4. What is the guard column in HPLC and what is its role?
The guard column is a short column that contains a same stationary phase as of the
analytical column.
But in the guard is filled with bigger particles size to avoid any unnecessary pressure. It removes the impurities present in the buffer and solvents of the mobile phase.
5. What is the HPLC column?
The HPLC column is the heart of the HPLC and it is responsible for the separation of different components. It is packed with the stationary phase like C18, C8 phase.
6. What is the mobile phase?
Mobile phase in HPLC is solvent or mixture of solvents or mixture of solvents containing solid buffers.
7. What is Isocratic mode of elution?
In the isocratic mode of elution composition of solvent in the mobile phase does not
change or remain constant. For example if the mobile phase is mixture of water and acetonitrile in a composition of 60:40. It means this composition will remain the same throughout the analysis.
8. What is the Gradient mode of elution?
In the gradient mode of composition of solvent changes with time. It is used to elute non-
polar compound.
9.What is RPC (Reverse phase chromatography)?
In RPC, the mobile phase is polar e.g., a mixture of Water/Buffer and organic solvents like
acetonitrile, methanol, ethanol, IPA, THF etc and the stationary phase is non-polar or less polar
e.g., C18 (ODS), C8, Cyno etc. The sample should be soluble in water or in a mixture of water and
organic solvents.
10. What are the different applications of HPLC?
The HPLC is used in both qualitative (identification test) and quantitative analysis in the following industries:
- Pharmaceuticals
- Food
- Testing Lab
- Research centres
- Biotech Industries
- Pesticide Industries
11. What are the different types of detectors used in HPLC?
The following detectors are used in HPLC analysis:
- Ultraviolet/Visible Absorbance (UV/Vis)
- Mass Spectrometer (MS)
- Refractive Index (RI)
- Evaporative Light Scattering (ELS)
- Fluorescence (FL)
- Electrochemical (EC)
12. What are the criteria for selecting a detector?
The selection of a Detector is based on:
Chemical nature of analytes and potential interferences
Limit of detection
Availability and/or cost of the detector
13. What are the advantages and disadvantages of RPC?
The following are the advantages and disadvantages of Reverse phase chromatography:
Advantages:
- Most commonly used chromatography
- Longer column life
- Lesser system equilibration time
- LC-MS compatible method can be developed
- Work well for the weak, acid, weak base and non-polar molecules
- Order of elution is hydrophobic to hydrophilic
Disadvantages:
Does not work for strongly ionized compounds e.g. Strong acidic compounds and strong Basic
compounds
14. Why is the mobile phase filtered out?
The following are the main reasons for filtering the mobile phase:
During the preparation of most of the mobile phase, solid chemicals like K2HPO4, KH2PO4, Na2HPO4, and NaH2PO4 etc. are used.
These solid chemicals are dissolved in water during buffer preparation.
These chemicals may contain water-insoluble particles as impurities and can cause problems during HPLC analysis such as noise, and column choking.
Secondly, during the preparation of the mobile phase, two or more solvents are mixed, and due to this mixing air also dissolves in the mobile phase.
N2 (Nitrogen) and O2 (Oxygent) in the air have UV absorption so they give their respective peaks as noise.
Dissolved air can also cause a drop in the pressure during analysis.
15. What is the general chapter of chromatography in USP?
The general chapter of chromatography in USP is <621>
16. Why Caffeine is used for the calibration of HPLC?
Caffeine is the stable molecule and readily available in pure form and that is why
caffeine is used for the calibration of HPLC.
17. How to decrease retention time in HPLC?
Increasing organic solvents such as methanol, and acetonitrile or decreasing aqueous
solvents such as water or buffer reduces retention time in reverse phase HPLC.
Decreasing nonpolar organic solvents or decreasing polar organic solvents reduces retention time in normal phase HPLC.
18. What causes HPLC retention time shifts?
Retention time may shift due to change due to the following reasons:
Change in the composition of the mobile phase
Change in the column temperature
Change in flow rate
Improper column equilibration
If bubbles trap in the tubing
19. Does temperature affect retention time in HPLC?
Yes. On increasing temperature retention time is decreased and on decreasing temperature
retention time is increased
20. Why does the HPLC retention time change for each run?
Due to pressure fluctuation or improper column equilibration retention time may change
in each run.